Currently, the major mode of biofuel production (production of fuel, such as ethanol, from biomass) is through direct fermentation, which accounts for 90% of the ethanol output in the U.S. (Licht, F. O. (2001) World Ethanol Markets, Analysis and Outlook, Kent, UK). Direct fermentation is the process in which a saccharolytic microorganism, such as yeast or bacteria, converts sugars to ethanol. These sugars may be simple (i.e. glucose) or complex (i.e. starch, cellulose, hemicellulose). Corn starch is the primary substrate used in ethanol producing plants today. One disadvantage of the use of corn starch in the direct fermentation production of ethanol is that corn is a component of many human and animal foods. Therefore, the use of the corn for the production of ethanol takes it out of the supply for human and animal consumption.
Other substrates, such as lignocellulosic biomass (i.e. grasses, small trees, paper waste, or saw dust), are also being researched for use in direct fermentation of biofuels. However, they also have limitations. Lignocellulose is comprised of cellulose, hemicellulose, pectin, and lignin, which require pretreatment processes to break down the biomass into its individual sugar components before microorganisms can utilize the substrate. This adds more cost in the areas of materials, plant design, and waste management. Furthermore, approximately 22-35% of the lignocellulose fraction is composed of lignin, which cannot be utilized by current direct fermentation approaches, and is discarded from the process as a recalcitrant waste material.
Another alternative method of biofuel production is indirect fermentation. Indirect fermentation is the process in which energy-rich gases which can provide an electron source, such as H2 and CO, and carbon sources, such as CO and CO2 are generated from carbon-containing, non-food agricultural and industrial waste material, and then are transferred to a bioreactor where anaerobic bacteria convert the gases to biofuels. The gases produced when lignocellulosic biomass is pyrolyzed (burned) would be an example of a type of waste gas that may be utilized in indirect fermentation. Synthesis gas (also referred to as “syngas”) (primarily CO, H2, and CO2) is a product of pyrolyzed biomass or coal and has been recognized for its potential role in the indirect fermentation of biomass to fuel alcohol (Zeikus, J. G, Annu Rev. Microbiol. 34:423-464 (1980). Another source of energy-rich waste gas is the basic oxygen steelmaking (LD converter) process, which produces a significant volume of waste gas consisting of 70% CO, 1-2% H2, and 10-15% CO2, which is also suitable for producing biofuels using the indirect fermentation process.
Anaerobic microorganisms such as acetogenic bacteria offer a viable route to convert waste gases to useful products, such as ethanol and n-butanol, via the indirect fermentation process. Such bacteria catalyze the conversion of H2 and CO2 and/or CO to biofuels with higher specificity, higher yields and lower energy costs than can be attained by the Fischer-Tropsch process, or other chemical biofuel production processes that use CO and H2. Several microorganisms capable of producing biofuels from waste gases and other substrates have been identified and are discussed below.
Six acetogenic bacteria have been described for use in the production of the biofuels, ethanol, n-butanol, or mixtures of these alcohols using at least one of the three main components of syngas (H2+CO2, or CO, or H2+CO2 and CO) as substrates. These include Butyribacterium methylotrophicum (Grethlein et al., 1990; Jain et al., 1994b), Clostridium ragsdalei (Huhnke et al., 2008), Clostridium carboxidivorans (Liou, et al., 2005), Moorella species HUC22-1 (Inokuma, et al., 2007), Clostridium autoethanogenum (Abrini et al., 1994), and Clostridium ljungdahlii (Arora et al, 1995; Batik et al., 1988; Batik et al. 1990; and Tanner et al., 1993). Of these representatives, only three—Clostridium ljungdahlii, Clostridium ragsdalei, and Clostridium autoethanogenum—are known to convert CO, or mixtures of CO and/or H2 and CO2 to acetic acid and ethanol. Thus, they are the only known organisms capable of forming a single alcohol (ethanol) end product, while simultaneously using all components of a synthesis gas stream. This group of bacteria, referred to in this document as the clostridial ethanologens, have significant commercial importance because the economics of the indirect fermentation process is advantaged by: (a) co-utilization of H2 and CO, so that the combined conversion rate exceeds 90%, and (b) production of a single alcohol, which permits the use of a simplified and less expensive biofuel recovery system.
Six clostridial ethanologens have been described in literature for the production of biofuels from synthesis gas:
(1) Clostridium ljungdahlii PETCT (ATCC No. 49587 and DSMZ No. 13528): This organism is the original type strain deposit for this species (Tanner et al., 1993). See U.S. Pat. No. 5,173,429.
(2) Clostridium ljungdahlii ERI-2 (ATCC No. 55380): The phylogenetic status of this organism remains unclear since it is apparently not identical to the PETC type strain, but is not included on the list of Approved Lists of Bacterial Names (Skerman et al., 1989). See U.S. Pat. No. 5,593,886.(3) Clostridium ljungdahlii C-01; (ATCC No. 55988): The phylogenetic status of this organism remains unclear since it is apparently not identical to the PETC type strain, but is not included on the list of Approved Lists of Bacterial Names (Skerman et al., 1989). See U.S. Pat. No. 6,136,577.(4) Clostridium ljungdahlii O-52; (ATCC No. 55989): The phylogenetic status of this organism remains unclear since it is apparently not identical to the PETC type strain, but is not included on the list of Approved Lists of Bacterial Names (Skerman et al., 1989). See U.S. Pat. No. 6,136,577.(5) Clostridium ragsdalei; (ATCC No. BAA-622): See U.S. Pat. No. US20080057554 (EP2061872A2).(6) Clostridium autoethanogenum; (DSMZ No. 10061) This isolate was described by Abrini et al., 1994 as producing ethanol and acetate from mixtures of CO, H2, and CO2.
In addition to patents describing the use of specific ethanologenic clostridia for biofuel production, as defined above, the following process patents exist for producing biofuels from waste gases such as synthesis gas. Certain patents also cover microorganisms known to produce multiple alcohols (primarily ethanol and n-butanol) from waste gases (e.g., Clostridium carboxidivorans):
(1) U.S. Pat. No. US20070275447A1 to Lewis et al. discloses Clostridium carboxidivorans ATCC No. BAA-624, a novel anaerobic clostridia bacterial species that is capable of synthesizing biofuels, including ethanol and n-butanol from waste gases such as synthesis gas.(2) U.S. Pat. No. 5,192,673 to Jain et al. discloses a mutant strain of Clostridium acetobytylicum and a process for making butanol with the strain.(3) U.S. Pat. No. 5,593,886 to Gaddy et al. discloses a process using Clostridium ljungdahlii ATCC No. 55380 for producing acetate and ethanol using waste gas (e.g. carbon black waste gas) as a substrate.(4) U.S. Pat. No. 5,807,722 to Gaddy et al. discloses a method and apparatus for converting waste gases into useful products such as organic acids and alcohols using anaerobic bacteria, such as Clostridium ljungdahlii ATCC No. 55380.(5) U.S. Pat. No. 6,136,577 to Gaddy et al. discloses a method and apparatus for converting waste gases into useful products such as organic acids and alcohols (particularly ethanol) using anaerobic bacteria, such as Clostridium ljungdahlii ATCC Nos. 55988 and 55989.(6) U.S. Pat. No. 6,136,577 to Gaddy et al. discloses a method and apparatus for converting waste gases into useful products such as organic acids and alcohols (particularly acetic acid) using anaerobic strains of Clostridium ljungdahlii. (7) U.S. Pat. No. 6,753,170 to Gaddy et al. discloses an anaerobic microbial fermentation process for the production of acetic acid.(8) U.S. Pat. No. 7,285,402 to Gaddy et al. discloses an anaerobic microbial fermentation process for the production of ethanol.
Despite the knowledge in the art regarding the use of microorganisms in the production of biofuels, there remains an ongoing need to discover and/or develop additional microorganisms that are capable of producing useful products such as biofuels using the indirect fermentation process. In particular, it would be advantageous to discover new ethanologenic clostridia that exhibit improved growth characteristics and high biofuel yields when cultivated under chemically-defined or low organic carbon growth conditions. Specifically, elimination of complex organic carbon sources such as yeast extract, beef extract, corn steep liquor, or soy tryptones from the bacterial growth medium could be considered desirable for production of biofuels via synthesis gas fermentation because (1) these components add additional expense to the biofuels production cost, and (2) complex organic carbon sources support foreign bacterial growth in the biofuels production fermentors, which negatively impacts biofuel yield and process economics.